Apparatus having rotatable planar trays for culturing microorganisms

ABSTRACT

Adherent or non-adherent microorganisms with or without microcarriers are cultured in an apparatus having rotatable planar trays that dip in and out of culture medium and momentarily retain culture medium due to rims along edges of the trays on both sides. The trays are in a housing that is substantially cylindrical and elongated along a longitudinal axis. The trays are arranged longitudinally to the longitudinal axis with spacing between the trays. The trays can be attached substantially radially to and around a shaft extending coaxially inside the housing, attached to both the shaft and the housing or attached to the housing without a shaft. Attachment is at one or two longitudinal edges of the trays. The shaft can be hollow and have a wall with openings. During culturing, the trays can be rotated in one direction 180° and then rotated through 180° in an opposite direction.

FIELD OF THE INVENTION

The invention relates to an apparatus for developing a reactive exchangesurface which is renewed regularly between a liquid phase and a gasphase. An apparatus of this type is intended, for example, for cellculture and the production of biological substances.

BACKGROUND OF THE INVENTION

In industries producing vaccines by means of cell culture or producingbiological substances secreted either by adherent or suspension cells orby micro-organisms, use is made of microcarriers, roller bottles, Rouxflasks, multiple chamber systems and fermentors.

Microcarriers are small microscopic beads on which adherent cells arecaused to grow. The beads are maintained in suspension in the culturemedium, hereafter called the liquid phase, with the help of a stirringsystem, the liquid phase being in contact with a gas phase. Everythingis contained in a tank called fermentor. This technique frequently usedin the vaccine industry is nonetheless confronted with two majordifficulties:

the difficulty of carrying out a suspension of micro-carriers in theculture medium which is compatible with anchorage and cell growthconditions,

the difficulty of ensuring the control and stability of the culturemedium pH, when taking into account the fact that the exchange surfacebetween the gas phase and the liquid phase is substantially limited.

Fermentors are also used for the culture of suspension cells ormicro-organisms. The handling difficulties are similar to thoseencountered during cultivation with a microcarrier; as the exchangesurface between the gas phase and the liquid phase is limited by thefermentor diameter, the pH stability becomes extremely difficult toensure.

Roller bottles are generally of cylindric shape and are designed to berotatable around their axes. The interior surfaces of these bottles areforeseen for cultivating adherent cells thereby forming culturesurfaces. The liquid of culture is introduced into the bottle togetherwith the cells. The rotating movement to which the bottles are subject,allows the culture surfaces to be covered with a film of medium therebyallowing cell growth on these culture surfaces. The culture surface istherefore limited to the size of the bottles. If the production of alarge quantity of cells is desired, a large number of bottles will benecessary. This is the case for industries producing, for example,interferon, insulin, viral vaccines, lymphokines. The handling of thesenumerous bottles during inoculation, medium change, virus introduction,supernatant and cell harvesting increases the risk of bottle and contentcontamination and requires the use of an important number of staff. Manyapparatus have been developed in order to increase the culture surfaceof roller bottles by increasing the surface of the bottle itself. Theseknown apparatus are intended for developing adherent cell culture butare not foreseen for providing a homogeneous suspension of microcarriersor of suspension cells.

Roux flasks have been used for more than a century for producingviruses, as well as other biological substances. They have the samedrawbacks as roller bottles, that is to say a limited culture surfaceper bottle and they require an important number of handling staff.

Document EP-A-0345415 discloses an apparatus in which the bottle has anenlarged surface. This enlargement of the surface is obtained byproviding the bottle body with a corrugated surface having corrugationswhich extend axially or longitudinally relative to the bottle axis. Thisbottle does not, however, allow liquid stirring. Therefore it does notpermit cultivation with microcarriers.

The surface used for cell development is also increased with theapparatus disclosed in document U.S. Pat. No. 3,839,155. With thisapparatus, additional surface is obtained with a parallel arrangement oftrays along the bottle axis. This apparatus however is not entirelysatisfactory for adherent cells because the trays, when in a horizontalposition, cannot retain a volume of liquid containing the cellsuspension. This causes the liquid to run out of the tray which does notfavour cell attachment during cultivation. Moreover, this apparatus isnot appropriate for the cultivation of so-called non-adherent cellsbecause nothing is provided here for stirring of the liquid phase.

Document LU-A-51646 describes a cell culture apparatus comprising Of aset of parallel trays placed one above the other so as to form culturechambers. All of this is placed inside the housing which can berotatable around an axis. This system permits cultivation of adherentcells on one side of the trays only. The interior surface of the housingis not used as a culture surface. Moreover, a stirring system allowingthe microcarriers or micro-organisms to be maintained in homogeneous andcontinuous suspension, cannot be introduced.

SUMMARY OF THE INVENTION

The present invention aims to remedy these drawbacks. To this end, thereis provided an apparatus comprising of an elongated closed housingdefining a volume for a reaction liquid, a device for introducing thereaction liquid into the housing and also for removing it therefrom andat least one set of trays arranged longitudinally inside the housingwith a regular spacing therebetween, the set of trays being mounted forrotation in such a way as be able to turn around a longitudinal axis,driving means to drive the set of trays in rotation in at least onedirection around the aforementioned longitudinal axis, the said trayshaving along their edges, rims which protrude from at least one of theopposite sides of the trays in such a way as, during the rotation of theaforementioned set of trays, the trays dip successively into thereaction liquid and each retain a predetermined quantity of liquidduring a part of their angular travel.

Therefore, owing to the layout of the proposed trays, according to theinvention, the exchange or culture surface is considerably increased forequal volumes and the Setting in suspension of non adherent cells,micro-organisms, microcarriers or microspheres is ensured without havingto use any internal stirring system. The rotation of the trays providesexcellent stirring of the liquid phase introduced into the apparatus bythe liquid being carried along by the rotating trays.

This stirring and the liquid movement within the housing which areobtained due to the rotation of the trays provides a homogeneousdistribution of the liquid phase elements and greatly increases theexchange and culture efficiency.

The trays can be laid out and fixed in various ways. In an exemplaryembodiment the trays are arranged radially around an axis which extendslongitudinally inside the housing and which are fixed to the said axisor to the internal surface of the housing or even both to the axis andthe internal surface of the housing. The trays are advantageously maderemovable and slidable for example into grooves provided for receivingtheir longitudinal edges. The sides of the trays can be corrugated,which further increases the development and yield of the exchange orculture surface.

Another interesting embodiment, because it is relatively easy toconstruct, comprises of a set of parallel trays assembled in such a wayas to present regular spacings between each Other. In this way also, thetrays can be fixed together to form a rotating body inside the housingor be mounted to the lining of the housing, for example by sliding theminto grooves provided on the interior surface of the housing.

Various other details of embodiment of the apparatus according to theinvention are defined in the subclaims.

In an advantageous manner, the invention provides rotating the traysalternately at least a half turn in one direction and then in the otherin order to obtain excellent wetting of the opposite sides of the trayswith a film of reaction agent.

The invention is described in further detail hereafter, by way ofexample, with reference to the appended drawings representing a fewpreferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents a perspective view, partially broken away, of a firstembodiment in accordance with this invention, in which the trays arearranged radially.

FIG. 2 represents on an enlarged scale, a cross-sectional view alongline II--II in FIG. 1.

FIG. 3 is a cross-sectional view of a second embodiment according to theinvention.

FIG. 4 is a partial cross-sectional view of a third embodiment accordingto the invention.

FIG. 5 is a perspective view of an exemplary tray of the apparatusaccording to FIG. 4.

FIG. 6 represents a cross-sectional view of a fourth embodimentaccording to the invention, in which the trays are fitted in parallelarrangement.

FIG. 7 is a longitudinal sectional view of the apparatus according toFIG. 6.

In the drawings, identical reference numerals designate parts orelements which are identical or similar and equivalent.

DESCRIPTION OF PREFERRED EMBODIMENTS

Generally, an apparatus according to this invention consists of anelongated closed housing which defines a volume for a liquid of reactionand a set of trays arranged longitudinally inside the housing with aregular spacing between them. The set of trays is mounted to be rotatedin such a way as to turn around a longitudinal axis thanks to anydriving means, known per se. Along their sides, the trays have raisedrims which protrude from one side or from the two sides of each tray insuch a way as to act as a retainer element for the liquid of reactioninto which the trays are dipping one after the other during theirrotation.

Herein after there will be described by way of example an apparatuscalled rotating bioreactor intended to allow the culture of adherent ornon adherent cells with or without microcarriers or the culture ofbacteria and other micro-organisms. To this end, if it is for adherentcell culture, for example, a liquid is introduced into the bioreactorhousing to breed and grow the cells and micro-organisms on the oppositesides of the trays and on the internal wall of the housing. In order toenhance culture performance, it is necessary to increase the availablesurface susceptible of entering into contact with the reactive liquidand equally to maximize its exploitation.

The exemplary embodiment illustrated in FIGS. 1 and 2 shows a rotatingbioreactor 10 in the shape of a flask comprising an external cylindricalhousing 11, mounted on rollers 15 which are to be driven into rotationaround its longitudinal axis 100. At its opposed ends, the housing 11has a base 16 and a neck 17. The neck has a bottle neck 18 which extendsconcentrically to the longitudinal axis 100 of the housing and whichserves to accommodate a closing means such as a screw cap 19. Inside thehousing, the trays 12 are arranged radially at regular intervals aroundan axis 20 which extends longitudinally inside the housing. The trays 12extend substantially the whole length of the housing up to apredetermined distance from the aforementioned base 16 and neck 17. Thetrays 12 may however be also shorter in length. These trays 12 whichhave a substantially rectangular shape, are normally rigid so that theycan support their own weight as well as the weight of the liquid whichthey are intended to retain as will be apparent later herin. Theirnumber can vary as a function of the technical characteristics of theapparatus.

In the embodiment illustrated in FIGS. 1 and 2, the trays extend fromthe axis 20 to the housing 11 and are fixed at their two opposite ends,respectively to the axis 20 and to the housing 11. To this end, theinner surface of the housing 11 and the outer surface of the axis 20have longitudinal grooves 14 and 21 respectively, arranged in such a wayas to securely fix the trays. The aforementioned grooves 14 and 21extend substantially the entire length of the housing 11 and of the axis20.

The lateral edges 28 and 29 of each tray 12 are equipped with raisedrims 31 and 32 which protrude from one side or from the two oppositesides of the tray. One portion of the lateral edge 29 intended to be atthe base of the housing 11 is provided to rest on the base 16 and to fitinto the radial grooves 24 provided in the inner side of the base 16.The other portion of the lateral edge 29 is curved and has a raised rim32 which protrudes from the plane of the tray. The curvature allows thetray and the adjacent end of the axis 20 to be aligned during theirmounting and provides a passage for the gases. The aforementioned raisedrims may possibly have a height which is decreasing from thelongitudinal side 26. Each tray together with the aforementioned raisedrims substantially form a basin which is able to retain temporarily adetermined quantity of the reactive liquid contained in the housing wheneach tray is dipping in turn in the reactive liquid during the entirerotation of the assembly and this through a portion of its ascendingangular path.

The axis 20 is advantageously hollow and has parallel longitudinalopenings 22 alternating with the grooves 21. The openings 22 allow thereactive liquid to flow inside the axis 20. During rotation of thetrays, the liquid then covers ribs 23 provided on the inner surface ofthe axis 20. The reactive liquid then flows over the ribs 23 below,crosses the corresponding openings 22 and flows along the trays 12underneath. Of course, the axis 20 could also be solid instead of beinghollow.

The trays generally have a planar surface. However, they can also have aslightly warped or out of true shape. With the aim of enlarging thetotal culture surface of each tray, the trays sides are provided withcorrugations, e.g. stripes. This further increases the development ofthe reaction or exchange surface and makes cell or micro-organismculture very efficient.

The inner surface of the housing 11 as well as each of the oppositesides 25a and 25b (see FIGS. 4 and 5) of the trays 12 are treated forcell culture either before assembling, during partial assembling or evenwhen the apparatus is completely assembled and this with the purpose ofobtaining an optimal surface treatment compatible with the nature of thesubstance or micro-organism to be stuck or cultivated.

When assembling the apparatus, before hermetically attaching the base 16to the housing 11, the trays 12 and the axis 20 are slid inside thehousing 11 so that the trays fit into the grooves 21 of the axis 20 andinto the longitudinal grooves 14 of the housing 11. The foregoing is adescription of one way of assembling the apparatus only. The trays canalso be fitted by sliding them into their respective grooves 14, 21, 24provided on the housing 11, on the axis 20 and on the base 16. Here itis the neck 17 which is hermetically attached to close the housing. Whenthe trays 12 have been placed inside the housing 11, it is then closedby attaching the neck 17.

The reaction agent and the substances to be cultivated are introducedthrough the bottle neck 18 of the housing placed, for example,vertically by means of a device, known per se, serving to introduce thereaction agent. The filling up of the housing can equally be done whensame is placed horizontally. In this case, it is one or more tubes (notshown in the drawings) placed in the neck extending either along theaxis of the housing 11 or along the inner surface of the neck whichallow liquids or gases to be introduced or drained or allow the contentsto empty the housing without having it to be opened.

In the example described above, the trays 12 are attached at their twolongitudinal edges. It must be noted that the trays 12 can be attachedat only one of their longitudinal edges, either by having one edge onlyattached to the axis 20, or by having one edge only attached to thehousing 11. In the first case the axis 20 and the trays 12 which itcarries form an assembly mounted in such a way as to be driven intorotation inside the housing. FIG. 3 represents an example of embodimentin which the trays 12 are attached to the axis 20 which, here, is asolid axis. The trays fit into grooves provided longitudinally in theexternal surface of the axis. This is mounted inside the housing 11supported by a framework 40. The free longitudinal edge of each tray 12has a longitudinal raised rim 33 which protrudes from the sides of thetray. The tray with the longitudinal raised rim 33 and theaforementioned lateral raised rims form a basin capable of retaining aquantity of reaction liquid as explained above. The axis 20 is coupledthrough a transmission device to a driving motor 15 so that the set oftrays can rotate around the longitudinal axis of the housing. The lattercould also accomodate several sets of trays capable of rotating aroundparallel axes.

In the case where the trays are solely attached to the housing, thelatter drives the trays into rotation around the longitudinal axis ofthe housing. FIG. 4 illustrates an example of embodiment in which thetrays are attached to the housing. The trays 12 are attached by means ofmortised dovetail shaped tongues 34 allowing to slidably receive thedovetail shaped tenon 35 formed along the external longitudinal edge 26of the tray 12. This way of attaching the trays proves very secure andparticularly appropriate when important masses of liquid need to beentrained along while ensuring an easy interchangeability of the trays,which can prove useful for replacing them in case of tray alteration.

On its opposite sides 25a and 25b, each tray 12 has a partition 36placed longitudinally along the free longitudinal edge 27 of the tray,possibly throughout the length of the tray 12. FIG. 5 shows aperspective view of such a tray layout. The longitudinal partition 36and the lateral rims 31 and 32 mentioned earlier herein substantiallyform with the tray a basin capable of momentarily retaining a determinedquantity of reaction liquid through the ascending angular path of thetray. In order to avoid that the free edges of the adjacent trays toucheach other in the event that the trays come to bend under the weight ofthe retained liquid or under the effect of vibrations, the longitudinalpartitions 36 have at their edge at least one protruding element 37which serves as a spacer and which provides a resting surface 38 for theedge 39 of the adjacent partition 36 placed on the adjacent tray 12,thereby to always ensure a spacing between two next adjacent trays andto allow the free passage of gases and the flow of liquids duringrotation of the trays.

The trays edges can also be of other shapes such as, for example, atleast one plain edge without any perpendicularly raised rim, an edgewith a lip or an edge for entirely resting on the base 16 of the housing11. The lateral rims and the longitudinal partitions have advantageouslythe same height in their adjoining area. The lateral rims have possiblya height which is decreasing regularly from the longitudinal edge 26 tothe longitudinal edge 27 of the tray.

When an appropriate quantity of liquid and gas is introduced into thehousing 11, the latter together with the trays 12 or the trays 12 aloneare driven into rotation around the longitudinal axis of the housingunder the action, of driving means 15 provided to this purpose. Duringtheir rotation, the trays 12 are dipping in turn into the reactionliquid and during the ascending travel of the trays, a determinedquantity of reaction liquid is retained by the rims of each tray and isentrained along with the trays. The liquid in excess flows graduallyover the rims and then through the openings 22 in the axis 20 or,possibly, through the space between two adjacent partitions 36 of nextadjacent trays (in the case of the embodiment of FIG. 4). The liquidfalls again to the lower part of the housing and covers all the surfaceslower down.

A rotation through at least 180° in one direction, followed by arotation through at least 180° in the opposite direction after return tothe starting point, allows a film of reaction agent to "wet" theopposite sides 25a, 25b of the trays 12 and the interior surfaces of thehousing and to cultivate adherent cells on these surfaces.

FIGS. 6 and 7 illustrate another way of arranging the trays. These aremounted parallel to each other following the direction of thelongitudinal axis of the housing and they are attached to the housing 11at their two opposite longitudinal edges.

The trays can also be attached to the housing 11 at one longitudinaledge only or attached together with a regular spacing therebetween toform a rotatable assembly. In the case where the trays are attached atone edge only, an advantageous arrangement consists in providing analternate attachment by successively fitting one longitudinal edge ofthe tray, then the opposite longitudinal edge of the next adjacent tray.The parallel arrangement of trays proves to be particularly simple toconstruct.

When adherent cells are cultivated within the housing, they can beharvested using conventional methods such as a proteolytic enzymesolution, e.g. trypsin, versen or any other chelating agent which aimsto detach cells from their support.

The rotating bioreactor is designed to allow equally culture of adherentcells as well as suspension cells. it also allows adherent cell culturewith micro-carriers. In this case, all the inner surfaces provided tothis end and the surface formed by the microcarriers can be used forcell growth.

Rotating the trays through half a turn in one direction and then in theother direction makes it possible to connect several apparatus togetherwhile working. To this end, an orientation mark is advantageouslyprovided on each apparatus, e.g. a reference mark, so as to ensure awell coordinated operation of the different apparatus. This allows, withthe help of tubes as mentioned above, to introduce or drain liquid atany time, to change medium without having to open the housing and totake representative microcarrier samples in order to observe cellgrowth.

The apparatus according to the invention permits two types of culture tobe carried out within a same bottle, viz adherent cell or micro-organismculture or non-adherent cell or micro-organism culture, with or withoutmicrocarriers. This significant and specific advantage of the inventionthus allows only one type of bottle to be used, whatever the type ofcells or micro-organisms cultivated may be.

The arrangement of several trays makes it possible to substantiallyincrease the culture surface for a constant volume. Thus, the apparatuspermits the production of a large quantity of cells in a restrictedtotal volume.

It is therefore apparent that the apparatus according to the inventionpermits both the culture surface and the exchange surface between theliquid and gas phases to be substantially increased within a givenvolume. The increase in surface as compared to a conventional rollerbottle or a fermentor can be higher than 1000%.

Another advantage of the apparatus according to the invention is thatthe culture conditions are kept independent from the size of thehousing, the ratio between the volume of liquid used, the volume of thegas phase and the total culture or exchange surface being constant. Thechangeover from a low capacity housing to a housing of a greatercapacity can therefore be done without modifying the parameters rulingthe culture.

The apparatus also permits a liquid phase to be concentrated byevaporation. The large exchange surface between the gas phase and theliquid phase allows, by circulating dry gas inside the housing or bysucking up the gas phase, to partially or entirely evaporate the liquidphase contained in the housing.

The embodiments of the invention described in the foregoing are examplesgiven by way of illustration and the invention is by no means limited tothese examples. Any modification, any variation and any equivalentarrangement should be considered as being comprised within the scope ofthe invention.

I claim:
 1. An apparatus for culturing microorganisms within a reactionliquid medium, comprising a closed elongated housing defining a volumeto receive a reaction liquid and a volume for a gas phase, the housingbeing comprised of a substantially cylindrical body elongated along alongitudinal axis and being closed at one end by a base and at the otherend by a closing cap for introducing the reaction liquid into thehousing and for removing it therefrom, a set of planar trays arrangedlongitudinally to said longitudinal axis inside the housing having theirlongitudinal edges attached to the housing and extending in parallel toone another with a regular spacing between them, the trays having alongtheir edges rims which protrude from both opposite planar sides of thetrays for at least momentarily retaining reaction liquid thereon,driving means to impart a rotating movement to the housing and set oftrays such that, when the set of trays is rotated through a path ofangular travel, the trays dip into the reaction liquid and retain apredetermined quantity of liquid momentarily on the planar sides of thetrays to cultivate cells thereon during a part of its angular travel. 2.The apparatus according to claim 1, wherein each tray is attached to thehousing at one of its longitudinal edge.
 3. The apparatus according toclaim 1,wherein the trays have corrugations on substantially the wholesurface of one of their opposite planar sides.
 4. The apparatusaccording to claim 1, wherein the trays are removably attached to aninner surface of the housing.
 5. A method of culturing microorganisms inan apparatus comprising a closed elongated housing defining a volume toreceive a reaction liquid and a volume for a gas phase, the housingbeing comprised of a substantially cylindrical body elongated along alongitudinal axis and being closed at one end by a base and at the otherend a closing cap for introducing the reaction liquid into the housingand for removing it therefrom, a set of trays arranged longitudinally tosaid longitudinal axis inside the housing having their longitudinaledges attached to the housing and extending in parallel to one anotherwith a regular spacing between them, the trays having along their edgesrims which protrude from both opposite planar sides of the trays for atleast momentarily retaining reaction liquid thereon, and driving meansto impart a rotating movement to the set of trays, said methodcomprising the steps of:(a) introducing a reaction liquid medium intosaid housing, (b) causing the housing and set of trays to rotate through180° in a first direction about the longitudinal axis of the elongatedhousing, (c) causing the housing and set of trays to rotate through 180°in a second direction opposite to said first direction about thelongitudinal axis of the elongated housing, whereby rotation of thetrays through 180° in alternate directions allows them to dip into thereaction liquid and retain a predetermined quantity of liquidmomentarily on each planar side of the trays in each direction ofrotation to thereby cultivate cells on both planar sides of the trays;and (d) repeating the steps (b) and (c).